Project Summary Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotes, include the receptors for acetylcholine, serotonin, GABA and glycine. They are involved in numerous neuropsychiatric, neurologic, and inflammatory diseases. All Cys-loop receptor family members in metazoans contain three structural domains: an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). The ECD and TMD are architecturally conserved between receptor families. The ICD is poorly conserved in both length and amino-acid composition and, for many subunits, contains large regions of predicted structural disorder. Due to the challenge of working with the ICDs of pentameric receptors, they have been essentially overlooked in terms of rigorous mechanistic characterization and direct exploration as pharmacological targets. The ICD is involved in finetuning plasma membrane expression levels, targeting, and function, in part mediated by protein-protein interactions (PPI) for example with chaperones. We envision that mechanistic knowledge of chaperone-mediated modulation will uncover new PPI drug targets. Through our published studies of the ICD we defined a linker that allowed for structure determination of the first in class structures of homo- and heteropentameric GABAA receptors. We also established that the ICD alone assembles into pentamers, establishing a novel role for the ICD in oligomeric assembly. In this competitive renewal we propose a multi- layered approach leveraging both soluble ICD chimeras and full-length receptors together with results obtained during the previous funding period in careful consideration of recently-published pLGIC structures. We discovered that the resistance to inhibitors of choline esterase (Ric-3) chaperone binds to a 24-amino acid L1- MX segment of 5-HT3A subunits. With the new proposed specific aims (SA) we will: (SA1) determine the role of the L1-MX segment in RIC-3 modulation of pLGIC assembly, (SA2) identify the segments within nAChR ICDs that mediate novel nAChR regulator (NACHO) modulation of pentameric assembly, and (SA3) characterize the mechanism by which opening of cation-conducting pLGICs involves translocation of the L1-MX segment through the MA-helix framed portals. In each aim, we will use biochemical and electrophysiological methods, coupled with overexpressed and purified proteins or proteins in their cellular environment. We anticipate that our studies will provide the basis for a novel class of targets for therapeutic development, PPI modulators for pentameric channel intracellular domains.